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. 1999 Aug 1;341(Pt 3):777–784. doi: 10.1042/0264-6021:3410777

Cytological evidence that the C-terminus of carnitine palmitoyltransferase I is on the cytosolic face of the mitochondrial outer membrane.

F R van der Leij 1, A M Kram 1, B Bartelds 1, H Roelofsen 1, G B Smid 1, J Takens 1, V A Zammit 1, J R Kuipers 1
PMCID: PMC1220418  PMID: 10417344

Abstract

Carnitine palmitoyltransferase I (CPT I) is a key enzyme in the regulation of beta-oxidation. The topology of this enzyme has been difficult to elucidate by biochemical methods. We studied the topology of a fusion protein of muscle-type CPT I (M-CPT I) and green fluorescent protein (GFP) by microscopical means. To validate the use of the fusion protein, designated CPT I-GFP, we checked whether the main characteristics of native CPT I were retained. CPT I-GFP was expressed in HeLa cells after stable transfection. Confocal laser scanning microscopy in living cells revealed an extranuclear punctate distribution of CPT I-GFP, which coincided with a mitochondrial fluorescent marker. Immunogold electron microscopy localized CPT I-GFP almost exclusively to the mitochondrial periphery and showed that the C-terminus of CPT I must be on the cytosolic face of the mitochondrial outer membrane. Western analysis showed a protein that was 6 kDa smaller than predicted, which is consistent with previous results for the native M-CPT I. Mitochondria from CPT I-GFP-expressing cells showed an increased CPT activity that was inhibited by malonyl-CoA and was lost on solubilization with Triton X-100. We conclude that CPT I-GFP adopts the same topology as native CPT I and that its C-terminus is located on the cytosolic face of the mitochondrial outer membrane. The evidence supports a recently proposed model for the domain structure of CPT I based on biochemical evidence.

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Selected References

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